This invention relates to novel muteins of growth hormone (xe2x80x9cGHxe2x80x9d), especially human growth hormone (xe2x80x9chGHxe2x80x9d), which diminish, decrease or inhibit the growth of animals or otherwise diminish, decrease or inhibit the effects of endogenous GH by acting as an antagonist to growth hormone receptors (xe2x80x9cGHRsxe2x80x9d). This invention also relates to DNAs encoding such muteins as well as methods for the treatment of diseases and disorders that are wholly or partially mediated by GHRs using a GH antagonist.
hGH and bovine growth hormone (xe2x80x9cbGHxe2x80x9d) are proteins of about 191 amino acids that are naturally synthesized in the anterior lobe of the pituitary. The molecular weight of the mature proteins is about 22,000 daltons, but they are initially made as pre-GHs with an extra 26 amino acids at the amino-terminus. This leader (or signal peptide) is-normally cleaved during secretion by pituitary cells to release the mature form.
Several forms of mature bGH have been found in nature. The amino-terminus can vary (due to variation in the site of cleavage during secretion) so that the mature protein begins with either NH2-Ala-Phe-Pro or NH2-Phe-Pro, the latter referred to as xe2x80x9c(des Ala) bGHxe2x80x9d. Additionally, the amino acid at bGH position 126 may be either Leu or Val, apparently as a result of allelic variation in the bovine population.
Exogenous administration of bGH to cattle increases milk production, feed efficiency, growth rate, and the lean-to-fat ratio, and decreases fattening time.
bGH has been produced by recombinant DNA techniques, see, e.g., Fraser, U.S. Pat. No. 4,443,539 (yeast); Buell, EP Appl. No. 103,395 (bacteria); Krivl, EP Appl. No. 193,515 (bacteria); Kopchick, EP Appl. No. 161,640 (encapsulated mouse cells implanted into animals); DeBoer, EP Appl. No. 75,444 (bacteria; gene modified to eliminate harmful secondary structure) and this has facilitated the production of analogues of bGH by site-specific mutagenesis. Thus, Aviv, GB No. 2,073,245 describes production of Met-Pro (des Ala) bGH, Met-Arg (des Ala) bGH, Met-Glu-Gly (des Ala) bGH, and des (Ala1-Phe2-Pro3-Ala4) bGH in E. coli. Brems et al., Proc. Natl. Acad. Sci. USA 85:3367-71 (1988) reported preparation of the bGH mutant K112L, which extended the hydrophobic face of the third alpha helix of bGH. The bGH (96-133) fragment of this mutant was also prepared.
The biological activity of proteolytic fragments of bGH has also been studied. Brems et al., Biochemistry 26:7774 (1987); Swislocki et al., Endocrinology 87:900 (1970); Paladini et al., TIBS 256 (Nov. 1979). The fragment bGH (96-133) is superior in growth-promoting assays to bGH (1-95) and bGH (151-191). Hara et al., Biochemistry 17:550 (1978); Sonenberg, U.S. Pat. Nos. 3,664,925 and 4,056,520; Chen and Sonenberg, J. Biol. Chem. 250:2510-14 (1977). An octapeptide derived from the amino-terminus of bGH has been shown to have hypoglycemic activity, see Ng et al., Diabetes 23:943-949 (1974), but it has no effect on growth. Similar results were observed with the fragment bGH (96-133). Graf et al., Eur. J. Biochem. 64:333-340 (1976); Hara et al., Biochem. 17:550-56 (1978).
Analogues of bGH have varied in growth-promoting activity, as have the known analogues of other GHs. However, a GH analogue having growth-inhibitory activity has not been previously been reported.
A variety of transgenic animals have been produced. Hammer et al., Nature 315:680-638 (1985) (rabbits, sheep and pigs). Certain of these animals have been caused to express a GH, and increased growth of such transgenic animals has been reported. Palmiter et al., Nature 300:611 (1982) microinjected the male pronucleus of fertilized mouse eggs with a DNA fragment containing the promoter of the mouse metallothionein I gene fused to the structural gene of rat GH. Several of the transgenic mice developed from the genetically modified zygote exhibited a growth rate substantially higher than that of control mice. (In effect, the genetically modified mouse serves as a test environment for determining the effect of the hormone on animal growth). Later, Palmiter et al., Science 222:809 (1983) demonstrated that a similar enhancement of growth could be obtained in transgenic mice bearing an expressible hGH gene. A like effect is observed when hGH releasing factor is expressed in transgenic mice. Hammer, et a., Nature 315:413 (1985).
hGH and bGH have also been expressed in transgenic animals. McGrane et al., J. Biol. Chem. 263:11443-51 (1988); Kopchick et al., Brazil. J. Genetics 12:37-54 (1989); Chen et al., J. Biol. Chem. 269:15892-97 (1994). However, transgenic animals characterized by an exogenous gene which confers a reduced growth phenotype were hitherto unknown.
Abnormally high GH levels have been associated with a number of disorders. The two classic disorders which are directly caused by high levels of GH are acromegaly and gigantism.
Changes associated with acromegaly include coarsening of body hair, thickening and darkening of the skin, enlargement and overactivity of sebaceous and sweat glands such that patients frequently complain of excessive perspiration and offensive body odor, overgrowth of the mandible, cartilaginous proliferation of the larynx causing a deepening of the voice, and enlargement of the tongue. In addition, excess GH in these patients is responsible for proliferation of articular cartilage which may undergo necrosis and erosion and endoneural fibrous proliferation which causes peripheral neuropathies. Excess GH also increases tubular reabsorption of phosphate and leads to mild hyperphosphatemia. Many of these symptoms are also seen in patients with gigantism.
The hallmark of treatments for acromegaly and giantism is their ability to lower insulin-like growth factor-1 (xe2x80x9cIGF-1xe2x80x9d) in plasma and/or tissue through either destruction of the pituitary or drug treatment. The role of IGF-1 in GH-mediated disorders, such as acromegaly and giantism is well recognized. Melmed et al., Amer. J. Med. 97:468-473 (1994).
The mainstay treatment modalities for these two disorders are pituitary ablation, radiation treatment, and bromocriptine mesylate. Pituitary ablation is a surgical procedure and, like any surgical procedure, is associated with a significant risk of complications including mortality. There are also risks associated with radiation treatment of the pituitary as well. In addition, the efficacy of radiation treatment may be delayed for several years. Moreover, these treatment modalities are not specific against that part of the pituitary that produces GH and may adversely affect adjacent tissue as well. Bromocriptine mesylate is a dopamine like drug which suppresses the production of GH. Recently, octreotide, a long-acting somatostatin analog has also been used to treat patients with acromegaly and gigantism which is refractory to surgery, radiation, and/or bromocriptine mesylate. Somatostatin inhibits the release of GH releasing hormone from the hypothalamus. GH releasing hormone stimulates production of GH in the pituitary and its secretion.
Another disorder that has been associated with abnormal GH levels is diabetes mellitus (DM). Characteristically, patients with poorly controlled DM have been found to have high levels of circulating GH. It has been shown that hypophysectomy could reduce diabetic hyperglycemia, thus strongly implicating the role of GH as an active component of the metabolic derangements of diabetes. Houssay and Biasotti, Rev. Soc. Argent. Biol. 6:251-296 (1930). It has been suggested that hypersecretion of GH may be the cause as much as the consequence of poor diabetic control. Press et al., New England J. Med. 310:810-814 (1984).
Most diabetics do not die of acute hyperglycemia. The overwhelming majority of diabetics die from complications associated with diabetes such as end organ failure. While diabetes affects almost all organs, heart and kidney failure are the most common causes of death. Other organs or systems that are commonly affected by DM are the eyes, the blood vessels and the nervous system. Patients with long standing diabetes will commonly have diabetic retinopathy, angiopathy and peripheral neuropathy. It is possible that normal GH secretion has a permissive role in patients predisposed to severe diabetic retinopathy. In such patients and in others in whom attempts to optimize glycemic control are unsuccessful, pharmacologic intervention may be beneficial not only in improving glycemic control but also in preventing severe proliferative diabetic retinopathy. Gerich et al., New England J. Med. 310:848-850 (1984).
Proliferative diabetic retinopathy is one of the leading causes of blindness in the United States and ranks second only to senile macular degeneration as a cause of permanent blindness. Benson et al., Diabetic Retinopathy, Duane, T., (eds.), Harper and Row, Philadelphia, Pa. pp. 1-24. In juveniles with insulin dependent diabetes, there is no evidence of diabetic retinopathy up to 5 years. However, 27% of juveniles who have had diabetes for 5 to 10 years have diabetic retinopathy. Also 71% of juveniles who have had diabetes for longer than 10 years have diabetic retinopathy. Greater than 90% of juveniles who have diabetes for 30 years will ultimately have diabetic retinopathy. Also, the 5 year mortality rate for individuals blind from diabetic retinopathy is 36%, in which death generally is caused by cardiac or kidney complications.
The pathogenesis of proliferative diabetic retinopathy is believed to be mediated by GH. It has been shown that human GH stimulates proliferation of human retinal microvascular endothelial cells in the diabetic; proliferation of these cells is the primary cause of proliferative diabetic retinopathy. Rymaszewski et al., Proc. Natl. Acad. Sci. USA 88:617-621 (1991). Thus, the involvement of GH in end organ damage in the diabetic is well established. Smith et al., Abstract of Presentation at ARVO meeting (May, 1995).
The kidneys are another organ that is affected by DM. Chen et al., Endocrinology 136:660-667 (1995). One type of pathology seen in patients with diabetic nephropathy is glomerulosclerosis. Glomerulosclerosis is the sclerosis of mesangial cells which is preceded by mesangial cell proliferation. Glomerular cells are responsible for filtering the blood and thus directly affect kidney function.
Transgenic mice which express bGH have been shown to have enlarged glomeruli which progressed to a state of glomerulosclerosis. Thus, GH has been implicated in the development of diabetic glomerulosclerosis. Doi et al., Am. J. Pathol. 137:541 (1990); Bell, Am. J. Med. Sci. 301:195 (1991).
The hypothesis that high levels of GH are responsible for many of the proliferative types of diseases seen in diabetics is further supported by the fact that dwarfs with diabetes do not develop the proliferative types of diseases seen in normal-sized diabetics. Merimee et al., New England J. Med. 298:1217-1222 (1978).
The present invention relates to proteins which are substantially homologous with a vertebrate GH but have growth-inhibitory activity.
We have discovered that mutation of Gly-119 in bGH to Arg (xe2x80x9cG119Rxe2x80x9d), Pro (xe2x80x9cG119Pxe2x80x9d), Lys (xe2x80x9cG119Kxe2x80x9d), Trp (xe2x80x9cG119Wxe2x80x9d) or Leu (xe2x80x9cG119Lxe2x80x9d), or the homologous GIy-120 in hGH to Arg (xe2x80x9cG120Rxe2x80x9d) or Trp (xe2x80x9cG120Wxe2x80x9d), results in a mutein (mutant protein or peptide fragment thereof) which has growth-inhibitory activity in vertebrates, especially mammals. These novel hormones may be administered to mammals (or other vertebrates), in particular humans and bovines, when growth inhibition is desirable.
In one embodiment of the invention, the hormone is produced exogenously and administered to the subject. In view of the size of the hormone, it is preferably produced by expression in a suitable host of a genecoding for it. Such a gene is most readily prepared by site-specific mutagenesis of a bGH gene. However, the hormone may also be produced by other techniques, such as by condensation of fragments of native bGH with a synthetic peptide carrying the replacement of amino acid. If a peptide fragment has the desired growth-inhibitory activity, it may be prepared in toto by a Merrifield-type synthesis.
In a second embodiment of the invention, this gene is introduced into a prenatal form of a mammal by known techniques, and the prenatal form is developed into a transgenic mammal which expresses a reduced growth phenotype. Conceivably, a mammal could be genetically modified after birth, i.e., xe2x80x9cgene therapyxe2x80x9d and/or xe2x80x9cgene/cell therapyxe2x80x9d.
Thus, growth-inhibited animals may be produced either by administration of the growth-inhibitory hormone of this invention in pharmaceutical form, or by genetic transformation of a prenatal or postnatal form of the animal.
The growth-inhibitory hormone, or the gene encoding it, is useful in the production of small animals for use in research facilities where space is restricted, as pets for pet lovers with limited quarters, and as livestock for farmers having small tracts.
The hormone may also be useful in the treatment of human gigantism, and in research on gigantism and dwarfism, in the treatment of diabetes and its sequelae, in the control of cholesterol, and in the prevention and treatment of certain cancers, particularly those whose growth is facilitated by GH or insulin-like growth factor-1 (xe2x80x9cIGF-1xe2x80x9d).
In general, GH antagonists are therapeutically or prophylactically useful in countering the adverse effects of elevated levels of GHs, both endogenous hormones and hormones administered clinically.
In the course of our work, we have discovered a correlation between the ability of mouse L cells to secrete the protein and the protein having an effect (positive or negative) on growth rate in a transgenic animal. The use of an L cell secretion assay to identify growth-modulating proteins is also a part of this invention.
Another aspect of the invention is to provide methods for the treatment of various diseases involving the production of excess GH, wherein the methods comprise the step of administering an effective amount of a GH antagonist. Specifically, the invention provides methods of treating acromegaly, gigantism, cancer, diabetes, vascular eye diseases (diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, retinopathy of sickle-cell anemia, etc.) as well as nephropathy.
Another aspect of the invention is to provide pharmaceutical formulations for the treatment of diseases, wherein the formulation comprise at least one GH antagonist. The formulations may be adapted for the treatment of specific diseases and adapted for the administration to specific body sites.
More specifically, the present invention relates to a method for treatment of disorders or diseases which are wholly or partially regulated by GHRs using an antagonist to GHRs. One example of the antagonist used in the present invention are proteins which are substantially homologous with a vertebrate GH but have growth-inhibitory activity. However, any antagonist of GHRs can be used in the method of the present invention.
The disorders or diseases which can be treated by the method of the present invention are many. Any disorder or disease that is exacerbated by the action of an agonist on GHRs can be treated by the present invention.
In one embodiment of the present invention, the disorder is acromegaly or gigantism. Specifically, the invention includes methods for treating acromegaly and/or giantism by administering to patients having such disorders a therapeutically effective amount of a growth hormone antagonist together with a pharmaceutically acceptable carrier to reduce a pathological effect or symptom of acromegaly and.or giantism and, in particular, to lower levels of IGF-1 in plasma and/or tissue. The pathological effects and symptoms of these disorders are discussed above.
In another embodiment, the disease is diabetes mellitus (DM). More specifically, the method of the present invention is used to prevent or reduce proliferative diseases associated with diabetes such as diabetic retinopathy and glomerulosclerosis in patients with DM. In particular, such methods include administering a growth hormone-antagonist in a therapeutically effective amount to reduce a pathological effect or symptom of diabetes, such as nephropathy or retinopathy, and/or to lower blood glucose levels.
Additionally,. GH is well known to possess anti-insulin or diabetogenic activities which involves the ability of GH to inhibit insulin""s action on target tissue, especially muscle and fat. This diabetogenic activity may result in an increase in the dose of insulin taken by the type I or type II diabetic patient. A GH antagonist may be used in this scenario to inhibit GH""s diabetogenic activity, thereby increasing a patient""s sensitivity to insulin. Thus, treatment of a diabetic patient with a GH antagonist could ultimately decrease the patient""s insulin requirement.
While GHs have not previously been implicated in hypercholesterolemia, in another embodiment, the method of the invention is used to lower serum cholesterol levels.
It is expected that the GH antagonists of the invention can reverse the anti-insulin effects observed in both type I and type II diabetes patients and have substantial clinical effects on diabetic control in patients. In type I diabetics, a rise in the serum glucose levels in the early hours of the morning while the patient remains asleep (the xe2x80x9cdawn phenomenaxe2x80x9d) has been linked to a nocturnal rise in GH levels. Treatment with a GH antagonist is expected to abolish this effect, leading to more consistent control and lower fasting serum glucose levels. In type II diabetics, the principal cause of elevated fasting glucose levels is unrestrained hepatic glucose production (xe2x80x9cHGPxe2x80x9d). In normal, non-diabetic subjects insulin effectively suppresses HGP to modest levels throughout the night. Type II patients, with insulin resistance at the level of the liver as well as in peripheral tissue, have greatly increased HGP despite normal or higher than normal insulin levels. Given the effects of GH in raising HGP and intensifying the insulin resistance of type II diabetic patients, it is expected that antagonism of GH action with a GH antagonist will have a significant effect in lowering HGP, resulting in a decrease in fasting glucose levels.
In another embodiment, the method of the present invention is used to treat or prevent cancers, including but not limited to, lymphoblastic leukemia, melanoma, lymphoma, adenocarcinoma, colorectalcarcinoma and lung, breast, ovarian, pancreatic and prostate cancer. Specifically, the, methods of the invention involve administering a growth hormone antagonist together with a pharmaceutically acceptable carrier to reduce tumor load and/or reduce a pathological effect or symptom of the cancer. Additionally, such methods decrease the need (frequency) for and improve the efficacy of radiation therapy and chemotherapy and generally improve the quality of life.
In other embodiments, the GH antagonists of the invention are used in methods for treating vascular eye diseases to reduce a pathological effect or symptom of the disease and/or prevent the development or retard the progression of neovascularization as found, for example, in diabetic retinopathy, retinopathy of prematurity, retinopathy of sickle-cell anemia and age-related macular degeneration.
In yet another embodiment, a GH antagonist is used to prevent restenosis after coronary balloon angioplasty.
In another embodiment, the present invention is used to counter the adverse effects of endogenous GH or clinically administered GH. Adverse effects of endogenous GH include but are not limited to symptoms associated with acromegaly, gigantism, and diabetes mellitus. These symptoms have been described in detail in the section on BACKGROUND OF THE INVENTION.
The appended claims are hereby incorporated by reference as a further enumeration of the preferred embodiments. All patents and publications cited in this specification are incorporated by reference.